Photosynthesis is vital for most life on Earth; however, many aspects of it remain poorly understood. The unicellular green alga Chlamydomonas reinhardtii has immense potential as a functional genomics platform for studying photosynthesis. Large numbers of Chlamydomonas mutants can easily be generated by random insertion of a drug resistance cassette, however, the traditional methods to map insertion sites are inefficient. We report a powerful tool, ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing), for high-throughput genotyping in a genome-wide collection of Chlamydomonas mutants. ChlaMmeSeq is also reproducible and quantitative, allowing quantification of relative abundances (thus growth rate) of individual mutants in pooled culture. ChlaMmeSeq opens the door to the generation of indexed mutant collections in green algae, enables genome-wide biological enrichment screens on plates, and allows quantitative growth rate measurement of pooled screens in photobioreactors. We anticipate that ChlaMmeSeq will facilitate the discoveries of novel components of photosynthesis and transform our understanding of photosynthesis.

Rajnish Khanna Abstract:

Guard cells respond to environmental and hormonal signals to control stomatal aperture. Opening and closing of stomatal pores provides an essential mechanism to regulate CO2 assimilation for photosynthesis balanced against transpirational water loss. Previously, Eisinger et al., 2012 (Mol. Plant 5: 601-610 and 716-725) reported that reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. In recent studies we have investigated how environmental and hormonal signals cause these rearrangements and found that COP1, a RING-finger-type ubiquitin E3 ligase, is required for destabilization and reorganization of the cytoskeleton necessary for stomatal closing, likely by the 26S proteasome. In addition to regulating the cytoskeleton, we discovered that COP1 function represses activity of S-type anion channels, which are also critical for stomatal closure. Thus, COP1 is a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function. Stomata are constitutively open in the absence of COP1 function. Treatment with a microtubule-destabilizing drug, oryzalin, leads to microtubule disassembly and stomatal closure in cop1 mutants. We are investigating whether guard cells pre-treated with oryzalin can recover by reassembling microtubule arrays and if so, whether cytoskeletal reorganization is required for stomatal reopening. We are employing the tools of genetics, cell biology, biochemistry and electrophysiology to address how guard cells move.